2'-Deoxy-2'-fluoro-beta-D-arabinonucleosides and oligonucleotides (2'F-ANA): synthesis and physicochemical studies

Recently, hybrids of RNA and D-arabinonucleic acids (ANA) as well as the 2'-deoxy-2'-fluoro-D-arabinonucleic acid analog (2'F-ANA) were shown to be substrates of RNase H. This enzyme is believed to be involved in the primary mechanism by which antisense oligonucleotides cause a reduction in target RNA levels in vivo. To gain a better understanding of the properties of arabinose based oligonucleotides, we have prepared a series of 2'F-ANA sequences of homopolymeric (A and T) and mixed base composition (A, T, G and C). UV thermal melting and circular dichroic (CD) studies were used to ascertain the thermodynamic stability and helical conformation of 2'F-ANA/RNA and 2'F-ANA/DNA hybrids. It is shown that 2'F-ANA has enhanced RNA affinity relative to that of DNA and phosphorothioate DNA. The 2'-fluoroarabino modification showed favorable pairing to single-stranded DNA also. This is in sharp contrast to ANA, which forms weak ANA/DNA hybrids at best. According to the measured thermodynamic parameters for duplex formation, the increased stability of hybrids formed by 2'F-ANA (e.g., 2'F-ANA/RNA) appears to originate from conformational pre-organization of the fluorinated sugars and a favorable enthalpy of hybridization. In addition, NMR spectroscopy revealed a five-bond coupling between the 2'F and the base protons (H6/H8) of 2'-deoxy-2'-fluoro-beta-D-arabinonucleosides. This observation is suggestive of a through-space interaction between 2'F and H6/H8 atoms. CD experiments indicate that 2'F-ANA/RNA hybrids adopt an 'A-like' structure and show more resemblance to DNA/RNA hybrids than to the pure RNA/RNA duplex. This feature is believed to be an important factor in the mechanism that allows RNase H to discriminate between 2'F-ANA/RNA (or DNA/RNA) and RNA/RNA duplexes.     

NMR study of the folding-unfolding mechanism for the thrombin-binding DNA aptamer d(GGTTGGTGTGGTTGG)

Hydrogen exchange rates of the imino protons of the thrombin-binding 15 mer DNA aptamer d(G(1)G(2)T(3)T(4)G(5)G(6)T(7)G(8)T(9)G(10)G(11)T(12)T(13)G(14)G(15)) in the presence of Sr(2+) were measured. In the temperature range 15-35 degrees C, the exchange rates of the eight iminos in the quadruplex core were not uniform, with the G(2), G(11) and G(15) iminos exchanging faster, the G(1), G(5), G(10) and G(14) iminos exchanging slower, and the G(6) imino exchanging at a medium rate. In the quadruplex G(1), G(5), G(10) and G(14) adopted syn glycosidic conformation, while G(2), G(6), G(11) and G(15) adopted anti-conformation. It was found that the four slowly exchanging iminos, which were all the syn-iminos, happened to be located in the TT loops that were not easy to open to the solvent. The anti-iminos exchanged faster, but the G(6) imino exchanged slower than other anti-iminos, because its hydrogen bond with the G(10)O6 was stabilized by the TGT loop. The fact that the G(6) imino exchanged at a faster rate than those syn-iminos in the TT loops suggested that the TGT loop was less stable than the TT loops. Unfolding mechanism for the quadruplex was thus proposed: The quadruplex first uncoupled the three base pairs: G(1)-G(15), G(2)-G(14) and G(5)-G(11), which were not protected by any loops. Then it opened the TGT loop. Finally, it opened the TT loops and the sequence became an unstructured random coil that exchanged with the quadruplex conformation. The conformational exchange between the quadruplex and random coil had been detected.     

A double chain reversal loop and two diagonal loops define the architecture of a unimolecular DNA quadruplex containing a pair of stacked G(syn)-G(syn)-G(anti)-G(anti) tetrads flanked by a G-(T-T) Triad and a T-T-T triple.

The architecture of G-G-G-G tetrad-aligned DNA quadruplexes in monovalent cation solution is dependent on the directionality of the four strands, which in turn are defined by loop connectivities and the guanine syn/anti distribution along individual strands and within individual G-G-G-G tetrads. The smallest unimolecular G-quadruplex belongs to the d(G2NnG2NnG2NnG2) family, which has the potential to form two stacked G-tetrads linked by Nn loop connectivities. Previous studies have focused on the thrombin-binding DNA aptamer d(G2T2G2TGTG2T2G2), where Nn was T2 for the first and third connecting loops and TGT for the middle connecting loop. This DNA aptamer in K(+) cation solution forms a unimolecular G-quadruplex stabilized by two stacked G(syn)-G(anti)-G(syn)-G(anti) tetrads, adjacent strands which are antiparallel to each other and edge-wise connecting T2, TGT and T2 loops. We now report on the NMR-based solution structure of the d(G2T4G2CAG2GT4G2T) sequence, which differs from the thrombin-binding DNA aptamer sequence in having longer first (T4) and third (GT4) loops and a shorter (CA) middle loop. This d(G2T4G2CAG2GT4G2T) sequence in Na(+) cation solution forms a unimolecular G-quadruplex stabilized by two stacked G(syn)-G(syn)-G(anti)-G(anti) tetrads, adjacent strands which have one parallel and one antiparallel neighbors and distinct non-edge-wise loop connectivities. Specifically, the longer first (T4) and third (GT4) loops are of the diagonal type while the shorter middle loop is of the double chain reversal type. In addition, the pair of stacked G-G-G-G tetrads are flanked on one side by a G-(T-T) triad and on the other side by a T-T-T triple. The distinct differences in strand directionalities, loop connectivities and syn/anti distribution within G-G-G-G tetrads between the thrombin-binding DNA aptamer d(G2T2G2TGTG2T2G2) quadruplex reported previously, and the d(G2T4G2CAG2GT4G2T) quadruplex reported here, reinforces the polymorphic nature of higher-order DNA architectures. Further, these two small unimolecular G-quadruplexes, which are distinct from each other and from parallel-stranded G-quadruplexes, provide novel targets for ligand recognition. Our results demonstrate that the double chain reversal loop connectivity identified previously by our laboratory within the Tetrahymena telomere d(T2G4)4 quadruplex, is a robust folding topology, since it has now also been observed within the d(G2T4G2CAG2GT4G2T) quadruplex. The identification of a G-(T-T) triad and a T-T-T triple, expands on the available recognition alignments for base triads and triples.     

Synthesis and properties of oligonucleotide chimeras containing 5'-amino-2'-deoxy-2'-fluoroarabinonucleosides

Oligonucleotide analogues comprised of 2'-deoxy-2'-fluoro-beta-D-arabinose units joined via P3'-N5' phosphoramidate linkages (2'F-ANA(5'N)) were prepared for the first time. Among the compounds prepared were a series of 2'OMe-RNA-[GAP]-2'OMe-RNA 'chimeras', whereby the "GAP" consisted of DNA, DNA(5'N), 2'F-ANA or 2'F-ANA(5'N) segments. The chimeras with the 2'F-ANA and DNA gaps exhibited the highest affinity towards a complementary RNA target, followed by the 5'-amino derivatives, i.e., 2'F-ANA > DNA > 2'F-ANA(5'N) > DNA(5'N). Importantly, hybrids between these chimeras and target RNA were all substrates of both human RNase HII and E. coli RNase HI. In terms of efficiency of the chimera in recruiting the bacterial enzyme, the following order was observed: gap DNA > 2'F-ANA > 2'F-ANA(5'N) > DNA(5'N). The corresponding relative rates observed with the human enzyme were: gap DNA > 2'F-ANA(5'N) > 2'F-ANA > DNA(5'N).     

The effect on quadruplex stability of North-nucleoside derivatives in the loops of the thrombin-binding aptamer

Modified thrombin-binding aptamers (TBAs) carrying uridine (U), 2'-deoxy-2'-fluorouridine (FU) and North-methanocarbathymidine (NT) residues in the loop regions were synthesized and analyzed by UV thermal denaturation experiments and CD spectroscopy. The replacement of thymidines in the TGT loop by U and FU results in an increased stability of the antiparallel quadruplex structure described for the TBA while the presence of NT residues in the same positions destabilizes the antiparallel structure. The substitution of the thymidines in the TT loops for U, FU and NT induce a destabilization of the antiparallel quadruplex, indicating the crucial role of these positions. NMR studies on TBAs modified with uridines at the TGT loop also confirm the presence of the antiparallel quadruplex structure. Nevertheless, replacement of two Ts in the TT loops by uridine gives a more complex scenario in which the antiparallel quadruplex structure is present along with other partially unfolded species or aggregates.     

Oligonucleotides comprised of alternating 2'-deoxy-2'-fluoro-beta-D-arabinonucleosides and D-2'-deoxyribonucleosides (2'F-ANA/DNA 'altimers') induce efficient RNA cleavage mediated by RNase H

Chimeric oligonucleotides comprised of alternating residues of 2'-deoxy-2'-fluoro-D-arabinonucleic acid (2'F-ANA) and DNA were synthesized and evaluated for an important antisense property-the ability to elicit ribonuclease H (RNase H) degradation of complementary RNA. Experiments used both human RNase HII and Escherichia coli RNase HI. Mixed backbone oligomers comprising alternating three-nucleotide segments of 2'F-ANA and three-nucleotide segments of DNA were the most efficient at eliciting RNase H degradation of target RNA, and were significantly better than oligonucleotides entirely composed of DNA, suggesting that these mixed backbone oligonucleotides may be potent antisense agents.     

Loop residues of thrombin-binding DNA aptamer impact G-quadruplex stability and thrombin binding

The thrombin-binding DNA aptamer (TBA) 5′-d(GGTTGGTGTGGTTGG)-3′ forms a G-quadruplex that is necessary for binding to the coagulation factor thrombin. The stability of the G-quadruplex of TBA when bound to thrombin and potassium ion (K⁺) were investigated for the wild-type oligonucleotide and for mutants in which thymine residues were substituted by adenine. In the presence of thrombin, G-quadruplexes formed by oligonucleotides in which the fourth or thirteenth residues were changed (T4A and T13A, respectively) were more unstable than that of wild-type, whereas T3A, T7A, T9A and T12A were more stable. The opposite effect was observed in the presence of 100 mM K⁺: the G-quadruplexes formed by T4A and T13A were more stable and T3A, T7A, T9A and T12A were more unstable than that of wild-type. Isothermal titration calorimetry measurements indicated that the binding constant of the interaction between T3A, T7A, T9A and T12A mutants and thrombin at 25 °C were close to that of wild-type, whereas T13A was significantly lower and T4A did not appear to bind to thrombin. Therefore, the stabilization of the G-quadruplex structure of TBA by thrombin appears to be due to an interaction between certain thymine nucleobases rather than to the quadruplex structure. The present study demonstrates that thrombin stabilizes the G-quadruplex via the interaction with residues in the loops but not via direct stabilization of G-quartets.     

Duplex recognition by oligonucleotides containing 2'-deoxy-2'-fluoro-D-arabinose and 2'-deoxy-2'-fluoro-D-ribose. Intermolecular 2'-OH-phosphate contacts versus sugar puckering in the stabilization of triple-helical complexes

To gain insight into the origins of the large binding affinity of RNA toward target duplexes, 2'-deoxy-2'-fluororibonucleic acid (2'F-RNA) and 2'-deoxy-2'-fluoroarabinonucleic acid (2'F-ANA) were tested for their ability to recognize duplex DNA, duplex RNA, and RNA-DNA hybrids. 2'F-RNA, 2'F-ANA, and the corresponding control single-stranded (ss) DNA strands were shown to form triple-helical complexes only with duplex DNA and hybrid DNA (Pu)-RNA (Py), but not with duplex RNA and hybrid RNA (Pu)-DNA (Py). In contrast, an RNA third strand recognized all four possible duplexes (DD, DR, RD, and RR) as previously demonstrated by Roberts and Crothers [(1992) Science 258, 1463-1466]. The 2'F-RNA (C3'-endo) strand exhibited significantly reduced affinity for duplexes compared to an unmodified RNA (C3'-endo) strand. These findings are consistent with the intermolecular 2'-OH-phosphate contact mechanism proposed by Escudé et al. [(1993) Nucleic Acids Res. 24, 5547-5553], as a ribo 2'-F atom should not interact with a negatively charged phosphate. In addition, they emphasize the role of the 2'-OH ribose as a general recognition and binding determinant of RNA. The 2'-F arabino modification (2'F-ANA, C2'-endo) led to a considerable increase in the binding affinity for duplex DNA, as compared to those of DNA and 2'F-RNA third strands. This is likely to be the result of a greater population of C2'-endo pucker of the 2'F-ANA compared to DNA. The enhancement observed for 2'F-ANA strands toward duplex DNA is comparable to that observed with 2'-OMe RNA. Since 2'F-ANA has been shown to be more resistant to nuclease degradation than DNA, these results are likely to stimulate experimental work on arabinose derivatives in laboratories concerned with targeting DNA sequences in vivo ("antigene" strategy).     

Heat capacity changes associated with guanine quadruplex formation: an isothermal titration calorimetry study

This study addresses the temperature dependence of the enthalpy of formation for several unimolecular quadruplexes in the presence of excess monovalent salt. We examined a series of biologically significant guanine-rich DNA sequences: thrombin binding aptamer (TBA) (d(G(2)T(2)G(2)TGTG(2)T(2)G(2)), PS2.M, a catalytically active aptamer (d(GTG(3)TAG(3)CG(3)T(2)G(2))), and the human telomere repeat (HT) (d(AG(3)(T(2)AG(3))(3))). Using CD spectra and UV melting, we confirmed the presence of quadruplex structures and established the temperature range in which quadruplex conformation is stable. We then performed ITC experiments, adding DNA to a solution containing excess NaCl or KCl. In this approach, only several additions are made, and only the enthalpy of quadruplex formation is measured. This measurement was repeated at different temperatures to determine the temperature dependence of the enthalpy change accompanying quadruplex formation. To control for the effect of nonspecific salt interactions during DNA folding, we repeated the experiment by replacing the quadruplex-forming sequences with a similar but nonfolding sequence. Dilution enthalpies were also subtracted to obtain the final enthalpy value involving only the quadruplex folding process. For all sequences studied, quadruplex formation was exothermic but with an increasing magnitude with increasing temperature. These results are discussed in terms of the change in heat capacity associated with quadruplex formation.     

The solution structure of d(G(4)T(4)G(3))(2): a bimolecular G-quadruplex with a novel fold

The G-rich 11-mer oligonucleotide d(G(4)T(4)G(3)) forms a bimolecular G-quadruplex in the presence of sodium ions with a topology that is distinct from the folds of the closely related and well-characterized sequences d(G(4)T(4)G(4)) and d(G(3)T(4)G(3)). The solution structure of d(G(4)T(4)G(3))(2) has been determined using a combination of NMR spectroscopy and restrained molecular dynamics calculations. d(G(4)T(4)G(3))(2) forms an asymmetric dimeric fold-back structure consisting of three stacked G-quartets. The two T(4) loops that span diagonally across the outer faces of the G-quartets assume different conformations. The glycosidic torsion angle conformations of the guanine bases are 5'-syn-anti-syn-anti-(T(4) loop)-anti-syn-anti in one strand and 5'-syn-anti-syn-anti-(T(4) loop)-syn-anti-syn in the other strand. The guanine bases of the two outer G-quartets exhibit a clockwise donor-acceptor hydrogen-bonding directionality, while those of the middle G-quartet exhibit the anti-clockwise directionality. The topology of this G-quadruplex, like other bimolecular fold-back structures with diagonal loops, places each strand of the G-quartet region next to a neighboring parallel and an anti-parallel strand. The two guanine residues not involved in G-quartet formation, G4 and G12 (i.e. the fourth guanine base of one strand and the first guanine base of the other strand), adopt distinct conformations. G4 is stacked on top of an adjacent G-quartet, and this base-stacking continues along with the bases of the loop residues T5 and T6. G12 is orientated away from the core of G-quartets; stacked on the T7 base and apparently involved in hydrogen-bonding interactions with the phosphodiester group of this same residue. The cation-dependent folding of the d(G(4)T(4)G(3))(2) quadruplex structure is distinct from that observed for similar sequences. While both d(G(4)T(4)G(4)) and d(G(3)T(4)G(3)) form bimolecular, diagonally looped G-quadruplex structures in the presence of Na(+), K(+) and NH(4)(+), we have observed this folding to be favored for d(G(4)T(4)G(3)) in the presence of Na(+), but not in the presence of K(+) or NH(4)(+). The structure of d(G(4)T(4)G(3))(2) exhibits a "slipped-loop" element that is similar to what has been proposed for structural intermediates in the folding pathway of some G-quadruplexes, and therefore provides support for the feasibility of these proposed transient structures in G-quadruplex formation.     

d(G3T4G4) forms unusual dimeric G-quadruplex structure with the same general fold in the presence of K+, Na+ or NH4+ ions

We have recently communicated that DNA oligonucleotide d(G(3)T(4)G(4)) forms a dimeric G-quadruplex in the presence of K(+) ions [J. Am. Chem. Soc.2003, 125, 7866-7871]. The high-resolution NMR structure of d(G(3)T(4)G(4))(2) G-quadruplex exhibits G-quadruplex core consisting of three stacked G-quartets. The two overhanging G3 and G11 residues are located at the opposite sides of the end G-quartets and are not involved in G-quartet formation. d(G(3)T(4)G(4))(2) G-quadruplex represents the first bimolecular G-quadruplex where end G-quartets are spanned by diagonal (T4-T7) as well as edge-type loops (T15-T18). Three of the G-rich strands are parallel while one is anti-parallel. The G12-G22 strand demonstrates a sharp reversal in strand direction between residues G19 and G20 that is accommodated with the leap over the middle G-quartet. The reversal in strand direction is achieved without any extra intervening residues. Here we furthermore examined the influence of different monovalent cations on the folding of d(G(3)T(4)G(4)). The resolved imino and aromatic proton resonances as well as (sequential) NOE connectivity patterns showed only minor differences in key intra- and interquartet NOE intensities in the presence of K(+), Na(+) and NH(4)(+) ions, which were consistent with subtle structural differences while retaining the same folding topology of d(G(3)T(4)G(4))(2) G-quadruplex.     

Solid-state 23Na NMR determination of the number and coordination of sodium cations bound to Oxytricha nova telomere repeat d(G4T4G4)

We report a solid-state (23)Na NMR study of the bound sodium cations in a G-quadruplex formed by Oxytricha nova telomere DNA repeat, d(G(4)T(4)G(4)) (Oxy-1.5). Using a 2D multiple-quantum magic-angle spinning (23)Na NMR method, we observed three sodium cations residing inside the quadruplex channel of the Na(+) form of Oxy-1.5. Each of these sodium cations is sandwiched between two G-quartets. We found no evidence for sodium cations in the T(4) loop region. For comparison, solid-state (15)N MAS NMR spectra were also obtained for the (15)NH(4)(+) form of Oxy-1.5. The insufficient resolution in the (15)N MAS NMR spectra did not permit determination of the number of NH(4)(+) ions inside the quadruplex channel. The solid-state (23)Na and (15)N NMR spectra for Oxy-1.5 were also compared with those obtained for guanosine 5'-monophosphate.     

Internal sodium ions and water molecules in guanine quadruplexes: magnetic relaxation dispersion studies of [d(G3T4G3)]2 and [d(G4T4G4)]2

The structural stability of guanine quadruplexes depends critically on an unusual configuration of dehydrated Na (+) or K (+) ions, closely spaced along the central axis of the quadruplex. Crystallography and NMR spectroscopy indicate that these internal ions can be located between the G-quartet planes as well as in the thymine loops, but the precise ion coordination has been firmly established in only a few cases. Here, we examine the bimolecular diagonal-looped foldback quadruplexes [d(G 3T 4G 3)] 2 (Q3) and [d(G 4T 4G 4)] 2 (Q4) by (2)H, (17)O, and (23)Na magnetic relaxation dispersion (MRD). The MRD data indicate that both quadruplexes contain Na (+) ions between the T 4 loops and the terminal G-quartets and that these ions have one water ligand. These ions exchange with external ions on a time scale of 10-60 mus at 27 degrees C, while their highly ordered water ligands have residence times in the range 10 (-8)-10 (-6) s. The MRD data indicate that Q4 contains three Na (+) ions in the stem sites, in agreement with previous solid-state (23)Na NMR findings but contrary to the only crystal structure of this quadruplex. For Q3, the MRD data suggest a less symmetric coordination of the two stem ions. In both quadruplexes, the stem ions have residence times of 0.6-1.0 ms at 27 degrees C. The equilibrium constant for Na (+) --> K (+) exchange is approximately 4 for both loop and stem sites in Q3, in agreement with previous (1)H NMR findings.     

NMR solution structure of a parallel LNA quadruplex

The solution structure of a locked nucleic acid (LNA) quadruplex, formed by the oligomer d(TGGGT), containing only conformationally restricted LNA residues is reported. NMR and CD spectroscopy, as well as molecular dynamics and mechanic calculations, has been used to characterize the complex. The molecule adopts a parallel stranded conformation with a 4-fold rotational symmetry, showing a right-handed helicity and the guanine residues in an almost planar conformation with three well-defined G-tetrads. The thermal stability of Q-LNA has been found to be comparable with that of [r(UGGGU)]₄, while a T_m increment of 20°C with respect to the corresponding DNA quadruplex structure [d(TGGGT)]₄ has been observed. The structural features of the LNA quadruplex reported here may open new perspectives for the biological application of LNAs as novel versatile tools to design aptamer or catalyst oligonucleotides.     

A new synthesis of 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)guanine (AraF-G)

Interesting and very promising antisense properties of 2'-deoxy-2'-fluoroarabinonucleic acids ((a) Wilds, C.J.; Damha, M.J. 2'-Deoxy-2'-fluoroarabinonucleosides and oligonucleotides (2'F-ANA): synthesis and physicochemical studies. Nucl. Acids Res. 2000, 28, 3625-3635; (b) Viazovkina, E.; Mangos, M.; Elzagheid, M.I.; Damha, M.J. Current Protocols in Nucleic Acid Chemistry 2002, 4.15.1-4.15.21) (2'F-ANA) has encouraged our research group to optimize the synthetic procedures for 2'-deoxy-2'-fluoro-beta-D-arabinonucleosides (araF-N). The synthesis of araF-U, araF-T, araF-A and araF-C is straightforward, (Tann, C.H.; Brodfuehrer, P.R.; Brundidge, S.P.; Sapino, C., Jr. Howell H.G. Fluorocarbohydrates in synthesis. An efficient synthesis of 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (beta-FIAU) and 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)thymine (beta-FMAU). J. Org. Chem. 1985, 50, 3644-3647; Howell, H.G.; Brodfuehrer, P.R.; Brundidge, S.P.; Benigni, D.A.; Sapino, C., Jr. Antiviral nucleosides. A stereospecific, total synthesis of 2'-fluoro-2'-deoxy-beta-D-arabinofuranosyl nucleosides. J. Org. Chem. 1988, 53, 85-88; Maruyama, T.; Takamatsu, S.; Kozai, S.; Satoh, Y.; Izana, K. Synthesis of 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine bearing a selectively removable protecting group. Chem. Pharm. Bull. 1999, 47, 966-970) however, the synthesis of the guanine analogue is more complicated and affords poor to moderate yields of araF-G (4) ((a) Elzagheid, M.I.; Viazovkina, E.; Masad, M.J. Synthesis of protected 2'-deoxy-2'-fluoro-beta-D-arabinonucleosides. Synthesis of 2'-fluoroarabino nucleoside phosphoramidites and their use in the synthesis of 2'F-ANA. Current Protocols in Nucleic Acid Chemistry 2002, 1.7.1-1.7.19; (b) Tennila, T.; Azhayeva, E.; Vepsalainen, J.; Laatikainen, R.; Azhayev, A.; Mikhailopulo, I. Oligonucleotides containing 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-adenine and -guanine: synthesis, hybridization and antisense properties. Nucleosides, Nucleotides and Nucl. Acids 2000, 19, 1861-1884). Here we describe an efficient synthesis of araF-G (4) that involves coupling of 2-deoxy-2-fluoro-3,5-di-O-benzoyl-alpha-D-arabinofuranosyl bromide (1) with 2-chlorohypoxanthine (2) to afford 2-chloro-beta-araF-I (3) in 52% yield. Nucleoside (3) was transformed into araF-G (4) by treatment with methanolic ammonia (150 degrees C, 6 h) in 67% yield.     

Selective recognition of parallel and anti-parallel thrombin-binding aptamer G-quadruplexes by different fluorescent dyes

G-quadruplexes (G4) have been found increasing potential in applications, such as molecular therapeutics, diagnostics and sensing. Both Thioflavin T (ThT) and N-Methyl mesoporphyrin IX (NMM) become fluorescent in the presence of most G4, but thrombin-binding aptamer (TBA) has been reported as the only exception of the known G4-forming oligonucleotides when ThT is used as a high-throughput assay to identify G4 formation. Here, we investigate the interactions between ThT/NMM and TBA through fluorescence spectroscopy, circular dichroism and molecular docking simulation experiments in the absence or presence of cations. The results display that a large ThT fluorescence enhancement can be observed only when ThT bind to the parallel TBA quadruplex, which is induced to form by ThT in the absence of cations. On the other hand, great promotion in NMM fluorescence can be obtained only in the presence of anti-parallel TBA quadruplex, which is induced to fold by K⁺ or thrombin. The highly selective recognition of TBA quadruplex with different topologies by the two probes may be useful to investigate the interactions between conformation-specific G4 and the associated proteins, and could also be applied in label-free fluorescent sensing of other biomolecules.     

Measured and calculated CD spectra of G-quartets stacked with the same or opposite polarities

Circular dichroism (CD) spectroscopy is widely used to characterize the structures of DNA G-quadruplexes. CD bands at 200-300 nm have been empirically related to G-quadruplexes having parallel or antiparallel sugar-phosphate backbones. We propose that a more fundamental interpretation of the origin of the CD bands is in the stacking interactions of neighboring G-quartets, which can have the same or opposing polarities of hydrogen bond acceptors and donors. From an empirical summation of CD spectra of the d(G)5 G-quadruplex and of the thrombin binding aptamer that have neighboring G-quartets with the same and opposite polarities, respectively, the spectra of aptamers selected by the Ff gene 5 protein (g5p) appear to arise from a combination of the two types of polarities of neighboring G-quartets. The aptamer CD spectra resemble the spectrum of d(G3T4G3), in which two adjacent quartets have the same and two have opposite polarities. Quantum-chemical spectral calculations were performed using a matrix method, based on guanine chromophores oriented as in d(G3T4G3). The calculations show that the two types of G-quartet stacks have CD spectra with features resembling experimental spectra of the corresponding types of G-quadruplexes.     

Favorable 2′-substitution in the loop region of a thrombin-binding DNA aptamer

Simple 2′-OMe-chemical modification in the loop region of the 15mer G-rich DNA sequence GGTTGGTGTGGTTGG is reported. The G-quadruplex structure of this thrombin-binding aptamer (TBA), is stabilized by single modifications (T?→?2′-OMe-U), depending on the position of the modification. The structural stability also renders significantly increased inhibition of thrombin-induced fibrin polymerization, a process closely associated with blood-clotting.     

Quadruplex formation as a molecular switch to turn on intrinsically fluorescent nucleotide analogs

Quadruplexes are involved in the regulation of gene expression and are part of telomeres at the ends of chromosomes. In addition, they are useful in therapeutic and biotechnological applications, including nucleic acid diagnostics. In the presence of K⁺ ions, two 15-mer sequences d(GGTTGGTGTGGTTGG) (thrombin binding aptamer) and d(GGGTGGGTGGGTGGG) (G3T) fold into antiparallel and parallel quadruplexes, respectively. In the present study, we measured the fluorescence intensity of one or more 2-aminopurine or 6-methylisoxanthopterin base analogs incorporated at loop-positions of quadruplex forming sequences to develop a detection method for DNA sequences in solution. Before quadruplex formation, the fluorescence is efficiently quenched in all cases. Remarkably, G3T quadruplex formation results in emission of fluorescence equal to that of a free base in all three positions. In the case of thrombin binding aptamer, the emission intensity depends on the location of the fluorescent nucleotides. Circular dichroism studies demonstrate that the modifications do not change the overall secondary structure, whereas thermal unfolding experiments revealed that fluorescent analogs significantly destabilize the quadruplexes. Overall, these studies suggest that quadruplexes containing fluorescent nucleotide analogs are useful tools in the development of novel DNA detection methodologies.     

NMR structure of the thrombin-binding DNA aptamer stabilized by Sr2+

The structure of thrombin-binding DNA aptamer complexed with a single Sr2+ ion (Sr2+:TBA complex) has been determined using NMR spectroscopy and restrained molecular dynamics simulations. The quadruplex structure for the Sr2+:TBA complex is similar in topology, but distinct in structure, from that previously reported for the K+:TBA complex. The inter-tetrad distance of the Sr2+:TBA complex is 3.8 angstroms, or 0.7 angstroms larger than in the K+:TBA complex. This substantial difference can be attributed to a different binding site for Sr2+ in the Sr2+:TBA complex than for K+ in the K+:TBA complex. The Sr2+:TBA complex assumes a 1:1 stoichiometry, and it is very likely that the Sr2+ ion simultaneously interacts with the eight O6 atoms of the two G-tetrads. The results indicate that quadruplex DNA structures are highly sensitive to the presence of specific metal ions. The binding of specific metal ions may modulate the biological activity of quadruplex DNA structures in vivo.